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R E S E A R C H Open AccessCirculating adenosine increases during human experimental endotoxemia but blockade of its receptor does not influence the immune response and subsequent organ

R E S E A R C H Open Access

Circulating adenosine increases during human experimental endotoxemia but blockade of its receptor does not influence the immune

response and subsequent organ injury

Bart P Ramakers1,2, Niels P Riksen1,3, Petra van den Broek1, Barbara Franke4, Wilbert HM Peters5,

Johannes G van der Hoeven2, Paul Smits1, Peter Pickkers2*

Abstract

Introduction: Preclinical studies have shown that the endogenous nucleoside adenosine prevents excessive tissue injury during systemic inflammation We aimed to study whether endogenous adenosine also limits tissue injury in

a human in vivo model of systemic inflammation In addition, we studied whether subjects with the common 34C > T nonsense variant (rs17602729) of adenosine monophosphate deaminase (AMPD1), which predicts

increased adenosine formation, have less inflammation-induced injury

Methods: In a randomized double-blinded design, healthy male volunteers received 2 ng/kg E Coli LPS

intravenously with (n = 10) or without (n = 10) pretreatment with the adenosine receptor antagonist caffeine (4 mg/kg body weight) In addition, lipopolysaccharide (LPS) was administered to 10 subjects heterozygous for the AMPD1 34C > T variant

Results: The increase in adenosine levels tended to be more pronounced in the subjects heterozygous for the AMPD1 34C > T variant (71 ± 22%, P=0.04), compared to placebo- (59 ± 29%, P=0.012) and caffeine-treated (53 ± 47%, P=0.29) subjects, but this difference between groups did not reach statistical significance Also the LPS-induced increase in circulating cytokines was similar in the LPS-placebo, LPS-caffeine and LPS-AMPD1-groups Endotoxemia resulted in an increase in circulating plasma markers of endothelial activation [intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM)], and in subclinical renal injury, measured by

increased urinary excretion of tubular injury markers The LPS-induced increase of these markers did not differ between the three groups

Conclusions: Human experimental endotoxemia induces an increase in circulating cytokine levels and subclinical endothelial and renal injury Although the plasma adenosine concentration is elevated during systemic

inflammation, co-administration of caffeine or the presence of the 34C > T variant of AMPD1 does not affect the observed subclinical organ damage, suggesting that adenosine does not affect the inflammatory response and subclinical endothelial and renal injury during human experimental endotoxemia

Trial Registration: ClinicalTrials (NCT): NCT00513110

* Correspondence: p.pickkers@ic.umcn.nl

2

Department of Intensive Care Medicine, Radboud University Nijmegen

Medical Center, Geert Grooteplein 10, 6500 HB, Nijmegen, The Netherlands

Full list of author information is available at the end of the article

© 2011 Ramakers et al.; licensee BioMed Central Ltd This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and

Sepsis, the systemic inflammatory response syndrome

that occurs during infection, is associated with

consider-able morbidity and mortality in non-cardiac intensive

care units [1] During sepsis, the initial inflammatory

response can be overwhelming, leading to significant

collateral damage to normal tissues

During systemic inflammation, the extracellular

con-centration of the endogenous nucleoside adenosine

increases rapidly [2,3], with concentrations increasing up

to 10-fold in septic shock patients [2] Animal studies

have shown that subsequent stimulation of adenosine

receptors, mainly the adenosine A2A receptor, on

var-ious immune cells potently reduces the inflammatory

response [4,5] In humans, however, evidence that

ade-nosine can limit the inflammatory response or prevent

tissue injury is limited [6]

Interestingly, a genetic loss-of-function variant of the

enzyme adenosine monophosphate deaminase (AMPD1)

was recently shown to improve prognosis in patients

with coronary artery disease [7], most likely because of

augmented adenosine formation during ischemia in

these patients [8] It is unknown whether subjects with

this polymorphism have an altered immune response or

whether these individuals are protected from

inflamma-tion-induced organ injury

In the present study, we addressed three major questions,

illustrated in Figure 1 First, does systemic inflammation

induced by experimental human endotoxemia increase the circulating adenosine concentration in vivo? Second, does this enhanced increase in circulating ade-nosine modulate the innate immune response? Third, does this increase reduce end-organ damage? We addressed these questions in healthy volunteers after systemic administration of lipopolysaccharide (LPS) with or without concomitant administration of the adenosine receptor antagonist caffeine In addition, we separately studied healthy volunteers with the 34C > T variant of the AMPD1 gene to test the third hypothesis (that is, that the inflammation-induced increase in circulating adenosine is augmented and organ damage

is attenuated in these subjects)

Materials and methods

Healthy volunteers

This study is registered at the ClinicalTrials.gov registry under the number NCT00513110 After the study was approved by the local ethics committee of the Radboud University Nijmegen Medical Centre, 43 healthy male volunteers provided written informed consent Since the inflammatory response that occurs in this particular model is different in females [9], we included male sub-jects only All volunteers had a normal physical exami-nation, electrocardiography, and routine laboratory values before the start of the experiment Since the prevalence of the AMPD1 SNP (single-nucleotide polymorphism) in Caucasian and African-American individuals is approximately 15% to 20%, we screened

a total of 43 individuals After genotyping of the AMPD1 rs17602729 variant (also known as 34C > T and Cys12Arg), we selected 10 subjects with the hetero-zygous (CT) genotype Of the remaining 33 subjects,

20 subjects (at random) were asked by an independent research nurse to participate in the study and were ran-domly assigned to either the control or caffeine-treated group Since the study was double-blind, the investiga-tors who were involved in the conduct of the study were not aware of whether the patient belonged to the AMPD1 group or the caffeine or placebo group (both without the AMPD1 polymorphism)

Volunteers were asked not to take any prescription drugs, and they refrained from caffeine intake 48 hours prior to the LPS administration The subjects were admitted to our clinical research unit on the day of the experiment and were kept under close observation for

10 hours

Experimental protocol

During the experiment, all volunteers were monitored for heart rate (electrocardiogram), blood pressure (intra-arterially), and body temperature (infrared tympanic thermometer; Sherwood Medical,‘s-Hertogenbosch, The

INNATEIMMUNITY (Cytokinerelease)

ORGANINJURY

ADENOSINE

Ͳ

Ͳ

AMPD1

CAFFEINE

+

Ͳ

Figure 1 Schematic view of the hypothesis During systemic

inflammation, the circulating adenosine concentration increases

rapidly, resulting in a negative feedback loop limiting (a)

inflammation-induced cytokine release and (b) tissue injury.

However, in the presence of caffeine, a non-selective adenosine

receptor antagonist, this mechanism of protection is lost and

inflammation-induced tissue damage will be aggravated In the

presence of the 34C > T variant of the AMPD1 gene, the

inflammation-induced increase in adenosine concentration is

augmented, and therefore the inflammatory response and organ

injury are reduced AMPD1, adenosine monophosphate deaminase.

Netherlands) from 2 hours before the administration of

LPS until the end of the experiment (8 hours after the

LPS administration) A cannula was inserted in a deep

forearm vein for prehydration (1.5 L of 2.5% glucose/

0.45% saline solution in the hour before LPS

administra-tion) and LPS infusion During the first 6 hours after

the LPS administration, all subjects received 150 mL/

hour and, after that period until the end of the

experi-ment, 75 mL/hour of 2.5% glucose/0.45% saline solution

to ensure an optimal hydration status [10]

An intra-arterial cannula was placed in the a

brachia-lis of the non-dominant arm, into which LPS was

injected at t = 0 hours The course of symptoms

(head-ache, nausea, shivering, and muscle and back pain) was

scored on a 6-point Likert scale (0 = no symptoms, 5 =

very severe symptoms), resulting in a total score of 0 to

25 Blood was collected at various time points after LPS

administration Furthermore, during the first 10 minutes

of every hour after LPS administration, forearm blood

flow was determined in both forearms with venous

occlusion plethysmography (Filtrass; DOMED

Medizin-technik GmbH, Munich, Germany) as previously

described [11,12]

The 20 subjects with the AMPD1 CC genotype (n =

20) received either caffeine (4 mg/kg body weight

intra-venously over 10 minutes [13]) or saline 10 minutes

before LPS infusion Caffeine, dosed at 4 mg/kg, has

been shown to effectively antagonize the hemodynamic

effects of adenosine, which are mediated by adenosine

A2A receptor stimulation [14] The 10 subjects

heterozy-gous for the AMPD1 polymorphism (CT genotype) also

received saline in a double-blinded fashion 10 minutes

before LPS infusion

Endotoxin

US Reference E coli endotoxin (Escheria coli O:113;

Clin-ical Center Reference Endotoxin, National Institutes of

Health, Bethesda, MD, USA) was used in this study Ec-5

endotoxin, supplied as a lypophilized powder, was

recon-stituted in 5 mL of 0.9% saline for injection and

vortex-mixed for at least 10 minutes after reconstitution The

endotoxin solution was administered as an intravenous

bolus injection at a dose of 2 ng/kg of body weight

Blood collection for adenosine measurement

The circulating adenosine concentration was measured

prior to and serially after the administration of LPS, as

previously described [15] With a special syringe system,

the blood was immediately mixed with a 2.5-mL

solu-tion containing pharmacological blockers of adenosine

formation, transport, and degradation immediately at

the tip of the syringe After blood was mixed with the

‘blocker solution’ and collected in the collection syringe

with a total volume of 5 mL, the hematocrit value was

determined in the mixture as a measure for dilution Afterward, blood samples were centrifuged for 10 min-utes at 1,000 g at 4°C and blood plasma was stored at -80°C until analyses

The‘blocker solution’ used to inhibit adenosine metabo-lism consisted of 40μM dipyridamole (adenosine trans-port inhibitor), 10μM erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) (adenosine deaminase inhibitor), 10μM iodotubericidine (ITU) (adenosine kinase inhibitor), 13.2

mM Na2EDTA (disodium ethylenediamine tetraacetate) (inhibits release from platelets and acts as a 5 ʹ-nucleoti-dase inhibitor), 118 mM NaCl, and 5 mM KCl

Genetic analysis

Blood was drawn in EDTA-containing vacutainers and stored at -80°C until DNA isolation Genomic DNA iso-lation was performed with a standard desalting protocol [16] Genotyping was performed by pyrosequencing according to the protocol of the manufacturer (Pyrose-quencing AB, now part of Qiagen GmbH, Hilden, Ger-many) [17], as previously described [8]

Determination of cytokines and adhesion molecules

Adhesion molecules ICAM (intercellular adhesion mole-cule) and VCAM (vascular cell adhesion molemole-cule), indi-cators of shedding from the endothelium, were used as markers of endothelial dysfunction To determine the concentration of the various cytokines and adhesion molecules, plasma was processed immediately by centri-fugation at 2,000 g at 4°C for 15 minutes and stored at -80°C until analyses Cytokine concentrations of tumor necrosis factor-alpha (TNF-a), interleukin (IL)-6, IL-1-receptor antagonist (IL1RA), and IL-10 were measured

in samples taken at baseline and at 30, 60, 120, 240, and

480 minutes after LPS administration and subsequently analyzed batch-wise with a Luminex assay (Luminex Corporation, Austin, TX, USA) [18]

Urine collection

Subjects collected urine in the 24 hours prior to the experiment During the experiment, urine was collected

2 hours prior to LPS administration, the first 3 hours after LPS infusion, and between 3 and 8 hours after LPS infusion During the sampling period, urine was kept on ice Urine was processed, and GSTA1-1 (glutathione transferase alpha 1-1) and GSTP1-1 (glutathione S-transferase pi 1-1), as markers of proximal and distal tubular injury, respectively, were measured as previously described [19]

Statistical analysis

Data with a Gaussian distribution were tested for signifi-cance by using repeated measures analysis of variance (ANOVA) Non-parametric data were analyzed with the

Friedman test The percentage increase in adenosine

concentrations and increase in GSTA1-1 and GSTP1-1

were analyzed with the paired Student t test Since most

of the data had a non-Gaussian distribution, data are

expressed as median (interquartile range [IQR]) unless

specified otherwise A P value of less than 0.05 was

con-sidered statistically significant

Results

Baseline characteristics

Demographic characteristics did not significantly

differ between the three groups of healthy volunteers

(Table 1)

Changes in clinical, inflammatory, and hemodynamic

parameters during human endotoxemia

In the 30 healthy volunteers, LPS administration

induced the expected influenza-like symptoms, such as

headache, nausea, and chills, starting after 60 to 120

minutes The symptoms were mild, and all volunteers

were symptom-free within 8 hours after LPS

administra-tion Peak symptoms occurred approximately 90

min-utes after LPS infusion Body temperature was

significantly elevated, with a peak temperature

approxi-mately 4 hours after LPS infusion (P < 0.0001, repeated

measures ANOVA for each group), and white blood cell

count decreased 1 hour after LPS administration, after

which there was an increase with a peak 8 hours after

LPS administration (P < 0.0001, repeated measures

ANOVA for each group) (Table 2) Plasma

concentra-tions of pro- and anti-inflammatory cytokines (TNF-a,

IL-6, IL-10, and IL1RA) are shown in Figure 2 Thus,

caffeine administration and the presence of the 34C > T

variant of the AMPD1 gene did not change the

inflam-matory response to LPS

LPS administration induced a decrease in blood

pressure and an increase in heart rate (Figure 3)

There were no significant differences in hemodynamic

parameters and plasma cytokine levels between the

three experimental groups Forearm blood flow

increased during experimental human endotoxemia,

with a maximal response 4 hours after LPS

administra-tion (Table 2)

The effect of lipopolysaccharide infusion on the endogenous adenosine concentration

The increase in adenosine levels tended to be more pro-nounced in the subjects heterozygous for the AMPD1 34C > T variant (from 9.0 [IQR 8.5 to 11.5] at baseline

to 16.5 [11.8 to 21.5] ng/mL 2 hours after LPS infusion,

an increase of 71% ± 22%; P = 0.04) compared with the placebo group (from 10.0 [IQR 8.8 to 13.0] at baseline

to 14.0 [12.3 to 19.0] ng/mL, an increase of 59% ± 29%;

P = 0.012), but this difference between groups did not reach statistical significance In the caffeine-treated sub-jects, the adenosine concentration increased from 12.0 [IQR10.0 to 18.0] at baseline to 18.0 [12.5 to 32.5] ng/

mL, an increase of 53% ± 47% (P = 0.29) Figure 4 illus-trates the LPS-induced changes in circulating adenosine Caffeine levels in the placebo and AMPD1 34C > T groups did not exceed 0.08 mg/mL either before or after LPS infusion In the caffeine group, caffeine levels were 0.04 [0.02 to 0.06] at baseline and 6.0 [5.6 to 6.4] mg/mL

1 hour after caffeine infusion (n = 10)

The effect of lipopolysaccharide infusion on end-organ injury

Vascular dysfunction

Plasma levels of ICAM and VCAM, markers of endothe-lial function, increased following LPS administration (Figure 5) (P < 0.0001 for ICAM and P = 0.006 for VCAM, ANOVA repeated measures) There was no sig-nificant difference in the LPS-induced increase in plasma ICAM and VCAM concentrations between the three groups (P > 0.1)

Renal injury

Glutathione-S-transferases (GSTs) are cytosolic enzymes that are present in the cells of the proximal tubule (GSTA1-1) and distal tubule (GSTP1-1) A very low urinary excretion rate is present during physiological circumstances Both GSTA1-1 and GSTP1-1 levels, respectively, increased during experimental endotoxemia (Figure 6) (n = 30, P < 0.0001) There were no differ-ences between the LPS-induced increase in the three experimental groups (P > 0.2)

Discussion

In the present study, we show for the first time that acute systemic inflammation induced by human experi-mental endotoxemia results in an increase in circulating endogenous adenosine in humans in vivo Apparently, the systemic inflammatory response during experimental endotoxemia is sufficient to stress the body to a level that induces adenosine release These results are in accordance with those of previous findings demonstrat-ing increased plasma adenosine concentrations in humans with septic shock [2,3,20] We found no evi-dence that circulating adenosine exerted immune

Table 1 Demographic characteristics

Experimental endotoxemia

(n = 10)

AMPD1 (n = 10)

Caffeine (n = 10) Age, years 23 (22-24) 23 (21-25) 22 (20-25)

Body mass index, kg/m2 21 (20-23) 23 (22-24) 22 (21-24)

Data for age and body mass index are presented as median (interquartile

Table 2 Clinical parameters and forearm blood flow response during human endotoxemia in the absence and

presence of caffeine or the AMPD1 polymorphism

Δ Temperature, °C Placebo 0.0 ± 0.0 0.3 ± 0.1 1.0 ± 0.1 1.3 ± 0.1 0.6 ± 0.1

AMPD1 0.0 ± 0.0 0.3 ± 0.1 1.0 ± 0.2 1.6 ± 0.2 0.9 ± 0.1 Caffeine 0.0 ± 0.0 0.3 ± 0.2 0.9 ± 0.2 1.6 ± 0.2 1.0 ± 0.2 Leukocytes, × 109/L Placebo 5.2 ± 0.8 3.0 ± 0.6 5.7 ± 0.6 8.9 ± 0.5 11.0 ± 0.5

AMPD1 5.1 ± 0.4 2.3 ± 0.2 6.4 ± 0.9 9.6 ± 1.1 11.9 ± 1.1 Caffeine 4.7 ± 0.3 2.4 ± 0.3 5.9 ± 0.7 10.6 ± 0.7 12.7 ± 0.7 FBF, mL/minute per dL forearm volume Placebo 2.8 (2.6-5.6) 5.3 (3.2-6.9) 3.8 (2.5-4.7) 7.3 (6.2-8.6) 6.4 (4.3-7.6)

AMPD1 3.1 (2.8-3.9) 3.1 (2.8-5.5) 3.0 (2.3-3.7) 6.2 (4.0-10.6) 5.8 (5.3-6.7) Caffeine 2.9 (2.1-3.5) 3.9 (3.1-4.7) 2.6 (2.2-3.0) 7.9 (5.3-10.7) 6.7 (5.7-7.4)

Lipopolysaccharide-induced changes were significant (P < 0.001, repeated measures analysis of variance) for each group but not significantly different between groups Data are presented as mean ± standard error of the mean Forearm blood flow (FBF) data are presented as median (interquartile range) since FBF data had a non-Gaussian distribution AMPD1, adenosine monophosphate deaminase.

TNF-Į

0

400

800

1200

1600

IL-6

0 400 800 1200 1600

Placebo Caffeine AMPD1

IL-10

0

0

50

100

150

200

250

Time (hrs) after LPS administration

IL1-RA

0 0 10000 20000 30000

Figure 2 Inflammatory parameters in the three groups (n = 10 per group) Administration of lipopolysaccharide (LPS) resulted in a marked increase in pro- and anti-inflammatory cytokines Data are expressed as median [nterquartile range]) and were analyzed with one-way analysis of variance (ANOVA) The probability values refer to the significant increase in circulating cytokines for each group, as analyzed with repeated measures ANOVA There was no significant difference between groups AMPD1, adenosine monophosphate deaminase; IL, interleukin; IL1RA, interleukin-1-receptor antagonist; TNF- a, tumor necrosis factor-alpha.

50

60

70

80

90

100

70 80 90 100

AMPD1 Caffeine

SBP

100

120

140

160

50 60 70 80 90

TimeafterLPSadministration(hrs)

Figure 3 Hemodynamic profile in response to endotoxemia (mean ± standard error of the mean, n = 10 subjects per group) Lipopolysaccharide (LPS) administration resulted in an increase in heart rate (HR) and decreases in mean arterial pressure (MAP), systolic blood pressure (SBP), and diastolic blood pressure (DBP) for each group (P < 0.01 repeated measures analysis of variance) There was no significant difference between groups AMPD1, adenosine monophosphate deaminase; bpm, beats per minute.

Time in hours after LPS administration

0

100

200

0 100 200

0 100 200

Figure 4 Percentage increase in plasma adenosine concentration after lipopolysaccharide (LPS) administration for each group Data are expressed as mean ± standard error of the mean Data were analyzed with the paired Student t test There were no significant differences between groups AMPD1, adenosine monophosphate deaminase.

modulatory effects or tissue-protective effects during inflammation Pretreatment with the adenosine receptor antagonist caffeine did not potentiate the inflammatory response or the inflammation-induced subclinical organ damage, suggesting that this increased adenosine con-centration does not act as a negative feedback signal to temper inflammation and organ damage in this model Previous in vitro and animal studies have provided robust evidence that endogenous adenosine plays a pivo-tal role in the limitation of excessive tissue injury in situations of inflammation, mainly by activation of ade-nosine A2Areceptor [5] In humans in vivo, however, data on the effect of inflammation on the endogenous adenosine concentration are limited to only one small study in which the plasma adenosine concentration was significantly higher in patients with septic shock com-pared with control patients [2]

In this study, we studied the effect of inflammation on circulating adenosine in a well-validated model of sys-temic inflammation [21] and used a previously described method to measure the plasma adenosine concentration [15] Our results show that, during endotoxemia, the endogenous adenosine concentration increases in time, with a maximum concentration reached 2 hours after LPS administration Recently, measuring circulating ade-nosine in 10 septic shock patients who were admitted to the intensive care unit, we found a median (IQR) adeno-sine concentration of 30.9 [24.1 to 39.8] ng/mL (BPR, NPR, PvdB, JGvdH, PS, and PP, unpublished observa-tions) The adenosine concentration was lower in the LPS-treated volunteers, probably indicating that the less

ICAM

0

Time (hrs) after LPS administration

200

300

400

500

100

200 300 400 500

100

Figure 5 Administration of lipopolysaccharide (LPS) resulted in a marked increase of intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), markers of endothelial activation Data are expressed as median [interquartile range] The

probability values refer to the significant increase in circulating adhesion molecules for each group, as analyzed with repeated measures analysis

of variance No significant difference between groups was found AMPD1, adenosine monophosphate deaminase.

GSTA1-1

0

200

400

600

800

1000

0

200

400

600

800

*

Day -1 3-8 hrs after LPS

*

*

*

*

PlaceboAMPD1Caffeine

Figure 6 Excretion of glutathione-S-transferases (GSTs) in urine.

Administration of lipopolysaccharide (LPS) resulted in a marked

increase in the urinary excretion of markers of proximal and distal

tubular damage Data are expressed as percentage increase in time

after LPS infusion (median [interquartile range]) Data were tested

with a paired Student t test *P < 0.05 No significant difference

between groups was found AMPD1, adenosine monophosphate

deaminase; GSTA1-1, glutathione S-transferase alpha 1-1; GSTP1-1,

glutathione S-transferase pi 1-1.

severe and shorter duration of the inflammatory

response during experimental endotoxemia induces a

smaller insult compared with septic shock In addition,

in septic shock patients, not only the inflammatory

response but also tissue hypoperfusion may play a role

in the formation of adenosine We subsequently aimed

to demonstrate that this increased circulating adenosine

could act as a negative feedback molecule, which

attenu-ates the inflammatory response and ameliorattenu-ates

end-organ dysfunction To this end, subjects were pretreated

with the nonselective adenosine receptor antagonist

caf-feine [22] in a dose previously shown to completely

block the cardiovascular effects of adenosine [13]

Sub-jects were asked to refrain from caffeine ingestion for

the 48-hour period prior to the experiment in order to

reveal any effects of adenosine receptor stimulation [23]

At the moment of LPS administration, the plasma

caf-feine concentration averaged 6.0 mg/L, which is a

con-centration previously shown to effectively antagonize

adenosine receptor stimulation [14,24] In more detail,

we recently showed that an intravenous dose of caffeine

of 4 mg/kg, similar to the dose of the present study,

completely blunted ischemic preconditioning, which is

mediated by adenosine receptor stimulation [13] In

addition, our group has demonstrated, in the past, that

caffeine in a plasma concentration of 5 mg/L

signifi-cantly antagonizes the hemodynamic effects of

adeno-sine administration [14]

Previous studies in animal models have shown that

caffeine is able to potentiate the production of

pro-inflammatory cytokines both in vitro [25,26] and in vivo

[27] and that caffeine exacerbates tissue injury during

inflammation [5,24] In contrast to these results, in our

human endotoxemia model, caffeine did not augment

the immune response nor did it increase (subclinical)

organ damage There are several potential explanations

for this finding First, endogenous adenosine may not

have an important anti-inflammatory potential in

humans in vivo However, this is not likely, given the

consistent findings in animal studies and isolated cell

studies and given the observation that administration of

exogenous adenosine can limit the IL-6 response during

human experimental endotoxemia [6] Second, the

lim-ited increase in adenosine in our model might not be

sufficient to induce significant anti-inflammatory effects

Recently, Soop and colleagues [28] demonstrated that

the administration of 40 μg/kg per minute adenosine

attenuated the release of the soluble RAGE (receptor for

advanced glycation end products) but was unable to

decrease the pro-inflammatory response Unfortunately,

no endogenous adenosine concentrations were measured

in that study, although it was speculated that blood

ade-nosine levels were at the submicromolar range Finally,

it needs to be realized that caffeine only blocks the

adenosine A1, A2A, and A2B receptors in the dose we used Therefore, stimulation of the adenosine A3 recep-tor, which also exerts anti-inflammatory potential, may have counteracted the pro-inflammatory effects of caf-feine [29-31] Specific adenosine subtype receptor antagonists are being developed but are not currently available for human use

We studied the effect, in a separate group of healthy volunteers, of the common 34C > T variant of the AMPD1 gene on the adenosine concentration and sub-clinical end-organ damage during endotoxemia In Cau-casians, approximately 20% of subjects are heterozygous for this variant allele, encoding a premature stopcodon, which results in a dysfunctional enzyme [32] AMPD catalyzes the intracellular conversion of AMP into IMP (inosine monophosphate) Subjects heterozygous for this variant allele appear to have a 50% reduction in enzyme activity [33] Interestingly, heterozygosity was recently associated with an improved cardiovascular prognosis in patients with coronary artery disease, probably because

of an increased conversion of AMP into adenosine with subsequent increased adenosine concentrations and sub-sequent organ protection during ischemia [8] Consider-ing the beneficial cardiovascular effects of adenosine receptor stimulation in subjects with AMPD deficiency [7,34], we hypothesized that endotoxemia-induced ade-nosine formation and subsequent adeade-nosine receptor sti-mulation would also be potentiated Although the LPS-induced increase in adenosine concentrations tended to

be most strongly potentiated in the AMPD1 heterozy-gous group (with a mean increase of 71% versus 59% and 53% in the placebo and caffeine groups, respec-tively), this difference between groups did not reach sta-tistical significance Moreover, we did not observe an attenuation of organ damage in subjects heterozygous for the AMPD1 variation A different route of adenosine formation during inflammation as compared with situa-tions of ischemia could be an explanation During ische-mia/hypoxia, an increased intracellular degradation of ATP significantly contributes to the increase in extracel-lular adenosine In this situation of increased intracellu-lar AMP availability, a reduction of AMPD activity could have an important effect on adenosine formation

In contrast, during inflammation, the main source of adenosine formation following endotoxemia is the extra-cellular hydrolysis of ATP instead of an intraextra-cellular increase in AMP Previous studies have suggested that inflammation directly leads to active release of adenine nucleosides, such as ATP, as well as passive release due

to endothelial cell damage [35] ATP is then quickly converted into adenosine During sepsis, tissue hypoxia will most likely also play an important role in the accu-mulation of adenosine [36,37]; however, this is unlikely during the relatively mild model of experimental

endotoxemia This could explain why the AMPD1

poly-morphism did not influence the inflammation-induced

increase in extracellular adenosine concentration The

lack of a significantly more pronounced increase in

cir-culating adenosine in AMPD1 subjects may also be

explained by the fact that adenosine is produced locally

in the tissue and the endothelium acts as an active

metabolic barrier for adenosine Thus, circulating

ade-nosine concentrations may not correctly reflect the

inflammation-induced adenosine increase in the

intersti-tial compartment Pharmacological interventions, such

as dipyridamole, an adenosine re-uptake inhibitor that

increases the local adenosine concentration [38], or

pen-toxifylline, of which the immunomodulatory effects

depend on sufficient levels of adenosine [20], may

repre-sent new therapeutic interventions to modulate the

immune response

Conclusions

Human experimental endotoxemia results in systemic

inflammation and increases the circulating endogenous

adenosine concentration Pharmacological blockade of

the adenosine receptors, however, does not augment the

innate immune response or its resultant (subclinical)

organ injury In addition, organ damage is not reduced

in subjects with the AMPD1 polymorphism, despite the

tendency to a more pronounced LPS-induced increase

in endogenous adenosine in these subjects Given these

observations, we conclude that, during human

endotox-emia, endogenous adenosine does not act as a negative

feedback molecule to limit the inflammatory response

and subsequent tissue injury

Key messages

• During human experimental endotoxemia (as a

model of systemic inflammation), the circulating

adenosine concentration increases

• Blockade of the adenosine receptor with caffeine

does not augment the inflammatory response or

subsequent organ damage

• The presence of the AMPD1 polymorphism is

associated with increased levels of adenosine but

does not affect the inflammatory response during

human experimental endotoxemia

• We conclude that the slight increase in

endogen-ous adenosine that occurs during human

endotoxe-mia is not sufficient to act as a negative feedback

mechanism to control the inflammatory response

Abbreviations

AMPD1: adenosine monophosphate deaminase; ANOVA: analysis of variance;

GSTA1-1: glutathione transferase alpha 1-1; GSTP1-1: glutathione

S-transferase pi 1-1; ICAM: intercellular adhesion molecule; IL: interleukin;

IL1RA: interleukin-1-receptor antagonist; IQR: interquartile range; LPS:

lipopolysaccharide; TNF- α: tumor necrosis factor-alpha; VCAM: vascular cell adhesion molecule.

Acknowledgements BPR is a recipient of an AGIKO fellowship of the Netherlands Organization for Scientific Research (ZonMw) The authors would like to thank Trees Jansen for her help with the cytokine measurements and Marlies Naber for her help with the genetic analysis.

Author details

1 Department of Pharmacology-Toxicology, Radboud University Nijmegen Medical Center, Geert Grooteplein 10, 6500 HB, Nijmegen, The Netherlands.

2 Department of Intensive Care Medicine, Radboud University Nijmegen Medical Center, Geert Grooteplein 10, 6500 HB, Nijmegen, The Netherlands.

3 Department of Internal Medicine, Radboud University Nijmegen Medical Center, Geert Grooteplein 10, 6500 HB, Nijmegen, The Netherlands.

4 Department of Human Genetics, Radboud University Nijmegen Medical Center, Geert Grooteplein 10, 6500 HB, Nijmegen, The Netherlands.

5 Department of Gastroenterology, Radboud University Nijmegen Medical Center, Geert Grooteplein 10, 6500 HB, Nijmegen, The Netherlands.

Authors ’ contributions BPR carried out the study, gathered all data, performed the statistical analysis and wrote the manuscript PvdB performed the adenosine and caffeine measurements BF supervised the genetic analyses and the writing

of the manuscript WHMP performed the GSTA1-1 and GSTP1-1 analyses PP, NPR, and PS supervised the conduct of the study and the writing of the paper JGvdH corrected the manuscript All authors read and approved the final manuscript.

Competing interests The authors declare that they have no competing interests.

Received: 3 June 2010 Revised: 1 October 2010 Accepted: 6 January 2011 Published: 6 January 2011

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doi:10.1186/cc9400 Cite this article as: Ramakers et al.: Circulating adenosine increases during human experimental endotoxemia but blockade of its receptor does not influence the immune response and subsequent organ injury Critical Care 2011 15:R3.

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